“The Ligands of CXCR4 in Vascularization” Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Trophologin Nancy Tuchscheerer aus Zwenkau, Deutschland Berichter: Universitätsprofessor Dr.rer.nat. Jürgen Bernhagen Universitätsprofessor Dr.techn. Werner Baumgartner Tag der mündlichen Prüfung: 14. Juni 2012 Die Dissertation ist auf den Internetseiten der Hochschulbibliothek online Verfügbar. The results of this work were in part published in: Liehn, E.A.*, N. Tuchscheerer *, I. Kanzler, M. Drechsler, L. Fraemohs, A. Schuh, R.R. Koenen, S. Zander, O. Soehnlein, M. Hristov, G. Grigorescu, A.O. Urs, M. Leabu, I. Bucur, M.W. Merx, A. Zernecke, J. Ehling, F. Gremse, T. Lammers, F. Kiessling, J. Bernhagen, A. Schober, and C. Weber. 2011. Double-edged role of the CXCL12/CXCR4 axis in experimental myocardial infarction. J Am Coll Cardiol . 58:2415-2423 Isabella Kanzler*, Nancy Tuchscheerer *, Sakine Simsekyilmaz, Simone Konschalla, Andreas Kroh, Guy Steffens, Andreas Schober, Christian Weber, Jürgen Bernhagen, Elisa A. Liehn. 2011. Macrophage migration inhibitory factor plays a pivotal role in EPCs-induced angiogenesis. Circ. Res. submitted . N. Tuchscheerer *, I. Kanzler*, Mark Vries, A. Wagenaar, E. Liehn, D.G.M. Molin, J. Bernhagen*, M.J. Post*. 2011. Macrophage migration inhibitory factor, a CXCR ligand with a function in arteriogenesis. Circ. Res. in preparation. TABLE OF CONTENTS Table of Contents TABLE OF CONTENTS ............................................................................................. I ABBREVIATIONS ...................................................................................................... V I INTRODUCTION .................................................................................................. 1 I.1 Cardiovascular Diseases, Myocardial Infarction, and Peripheral Artery Disease ........................................................................................................ 1 I.2 Angiogenesis and Arteriogenesis, Two Forms of Vascularization ............... 2 I.2.1 Angiogenesis ..................................................................................... 2 Mechanisms of Angiogenesis ....................................................... 3 Angiogenesis in Hypoxic Conditions ............................................. 4 Murine Myocardial Infarction Model, a Model of Angiogenesis ..... 6 The Role of Endothelial Progenitor Cells ...................................... 7 Endothelial Progenitor Cells in Angiogenesis and Vascular Repair ........................................................................................... 8 I.2.2 Arteriogenesis ................................................................................... 9 Stages of Arteriogenesis ............................................................. 11 The Role of Monocytes/Macrophages in Arteriogenesis ............. 12 Hindlimb Ischemia Model, a Model of Arteriogenesis.................. 16 I.2.3 Angiogenesis vs. Arteriogenesis ..................................................... 17 I.3 Cytokines and Chemokines ....................................................................... 18 I.3.1 Receptor / Ligand Interactions in Monocyte Recruitment ................ 20 I.3.2 CXCR4: Structure, Functions and Ligands ...................................... 21 Stromal Cell-derived Factor-1, CXCL12 ...................................... 21 The CXCL12 / CXCR4 Axis in Myocardial Infarction ................... 22 Macrophage Migration Inhibitory Factor ...................................... 23 I.3.3 The Role of Chemokines and Cytokines in Myocardial Infarction and Peripheral Artery Disease......................................................... 27 I.3.4 The Role of Chemokines and Cytokines in Endothelial Progenitor Cells Function.................................................................................. 28 II AIM OF THE STUDY ......................................................................................... 30 I TABLE OF CONTENTS III MATERIAL AND METHODS ............................................................................. 31 III.1 General Equipment .................................................................................... 31 III.2 General Solutions, Media and Buffers ....................................................... 32 III.3 Chemokines/Cytokines and Recombinant Proteins ................................... 33 III.4 Antibodies .................................................................................................. 34 III.4.1 Primary Antibodies .......................................................................... 34 III.4.2 Isotype Controls .............................................................................. 34 III.4.3 Secondary Antibodies ..................................................................... 34 III.4.4 Directly Conjugated Antibodies ....................................................... 34 III.4.5 Blocking Antibodies ......................................................................... 35 III.5 Mice ........................................................................................................... 35 III.6 Cell culture and Cell Isolation .................................................................... 36 III.6.1 Cell Culturing of WEHIs and SVECs ............................................... 36 III.6.2 Isolation of Angiogenic Early Outgrowth EPCs ............................... 36 III.6.3 Cardiomyocyte Isolation and Hypoxia Experiments ........................ 37 III.6.4 Isolation of Blood Monocytes .......................................................... 37 III.6.5 Isolation of Bone Marrow-Derived Monocytes ................................. 37 III.7 Biomolecular Methods ............................................................................... 38 III.7.1 mRNA Isolation and Reverse Transcription - Polymerase Chain Reaction .......................................................................................... 38 III.7.2 Quantification of RNA and Quantitative Real-Time Polymerase Chain Reaction .......................................................................................... 39 III.8 Protein Assays ........................................................................................... 41 III.8.1 Flow Cytometry Analysis ................................................................. 41 III.8.2 Histochemistry and Immunohistochemistry ..................................... 42 Histomorphometry and Determination of Myocardial Infarction Size ............................................................................. 42 Cellular Stainings of Inflammatory Cells in the Heart .................. 43 Cellular Stainings of Receptors and MIF in Hindlimb Tissue ....... 43 III.8.3 AcLDL and Lectin Uptake ................................................................ 44 III.9 Functional Assays ...................................................................................... 44 III.9.1 Chemotaxis Assay ........................................................................... 44 III.9.2 Transmigration Assay ...................................................................... 44 III.9.3 Static Cell Adhesion Assay ............................................................. 45 II TABLE OF CONTENTS III.9.4 Flow Cell Adhesion Assay ............................................................... 45 III.9.5 Matrigel Assay in vitro ..................................................................... 46 III.9.6 Electron Microscopy ........................................................................ 46 III.9.7 Lipid Extraction and HPLC .............................................................. 46 III.9.8 Gas-Chromatography of Fatty Acids ............................................... 47 III.10 Animal Experiments ................................................................................... 47 III.10.1 Murine Model of Myocardial Infarction ............................................. 47 III.10.2 Bone Marrow Reconstitution ........................................................... 48 III.10.3 Ultrasound Analysis ......................................................................... 48 III.10.4 Langendorff Perfusion ..................................................................... 48 III.10.5 Vessel Density (High-Resolution Computer Tomography) .............. 49 III.10.6 Ischemia Hindlimb Model ................................................................ 49 III.10.7 Pre- and Post Laser Doppler Measurements .................................. 50 III.11 Statistical Analysis ..................................................................................... 51 IV RESULTS .......................................................................................................... 5251 IV.1 The Role of the CXCL12 / CXCR4 Axis in Experimental Myocardial Infarction .................................................................................................... 52 IV.1.1 Analysis of MI Size and Inflammatory Cell Content ......................... 52 IV.1.2 Analysis of Cardiac Function after MI .............................................. 55 IV.1.3 The Role of CXCR4 for EPC Trafficking and Function .................... 60 IV.1.4 Myocardial Apoptosis after Myocardial Infarction ............................ 62 IV.1.5 Electron Microscopy